Treprostinil administration by inhalation

ABSTRACT

Treprostinil can be administered using a metered dose inhaler. Such administration provides a greater degree of autonomy to patients. Also disclosed are kits that include a metered dose inhaler containing a pharmaceutical formulation containing treprostinil.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation of U.S. application Ser. No.11/748,205, filed May 14, 2007, which claims priority to U.S.provisional application No. 60/800,016 filed May 15, 2006, which areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present application relates to methods and kits for therapeutictreatment and, more particularly, to therapeutic methods involvingadministering treprostinil using a metered dose inhaler and relatedkits.

BACKGROUND OF THE INVENTION

All blood is driven through the lungs via the pulmonary circulation inorder, among other things, to replenish the oxygen which it dispenses inits passage around the rest of the body via the systemic circulation.The flow through both circulations is in normal circumstances equal, butthe resistance offered to it in the pulmonary circulation is generallymuch less than that of the systemic circulation. When the resistance topulmonary blood flow increases, the pressure in the circulation isgreater for any particular flow. The above described condition isreferred to as pulmonary hypertension (PH). Generally, pulmonaryhypertension is defined through observations of pressures above thenormal range pertaining in the majority of people residing at the samealtitude and engaged in similar activities.

Pulmonary hypertension may occur due to various reasons and thedifferent entities of pulmonary hypertension were classified based onclinical and pathological grounds in 5 categories according to thelatest WHO convention, see e.g. Simonneau G., et al. J. Am. Coll.Cardiol. 2004; 43(12 Suppl S):5S-12S. Pulmonary hypertension can be amanifestation of an obvious or explicable increase in resistance, suchas obstruction to blood flow by pulmonary emboli, malfunction of theheart's valves or muscle in handling blood after its passage through thelungs, diminution in pulmonary vessel caliber as a reflex response toalveolar hypoxia due to lung diseases or high altitude, or a mismatch ofvascular capacity and essential blood flow, such as shunting of blood incongenital abnormalities or surgical removal of lung tissue. Inaddition, certain infectious diseases, such as HIV and liver diseaseswith portal hypertension may cause pulmonary hypertension. Autoimmunedisorders, such as collagen vascular diseases, also often lead topulmonary vascular narrowing and contribute to a significant number ofpulmonary hypertension patients. The cases of pulmonary hypertensionremain where the cause of the increased resistance is as yetinexplicable are defined as idiopathic (primary) pulmonary hypertension(iPAH) and are diagnosed by and after exclusion of the causes ofsecondary pulmonary hypertension and are in the majority of casesrelated to a genetic mutation in the bone morphogenetic proteinreceptor-2 gene. The cases of idiopathic pulmonary arterial hypertensiontend to comprise a recognizable entity of about 40% of patients caredfor in large specialized pulmonary hypertension centers. Approximately65% of the most commonly afflicted are female and young adults, thoughit has occurred in children and patients over 50. Life expectancy fromthe time of diagnosis is short without specific treatment, about 3 to 5years, though occasional reports of spontaneous remission and longersurvival are to be expected given the nature of the diagnostic process.Generally, however, disease progress is inexorable via syncope and rightheart failure and death is quite often sudden.

Pulmonary hypertension refers to a condition associated with anelevation of pulmonary arterial pressure (PAP) over normal levels. Inhumans, a typical mean PAP is approximately 12-15 mm Hg. Pulmonaryhypertension, on the other hand, can be defined as mean PAP above 25mmHg, assessed by right heart catheter measurement. Pulmonary arterialpressure may reach systemic pressure levels or even exceed these insevere forms of pulmonary hypertension. When the PAP markedly increasesdue to pulmonary venous congestion, i.e. in left heart failure or valvedysfunction, plasma can escape from the capillaries into the lunginterstitium and alveoli. Fluid buildup in the lung (pulmonary edema)can result, with an associated decrease in lung function that can insome cases be fatal. Pulmonary edema, however, is not a feature of evensevere pulmonary hypertension due to pulmonary vascular changes in allother entities of this disease.

Pulmonary hypertension may either be acute or chronic. Acute pulmonaryhypertension is often a potentially reversible phenomenon generallyattributable to constriction of the smooth muscle of the pulmonary bloodvessels, which may be triggered by such conditions as hypoxia (as inhigh-altitude sickness), acidosis, inflammation, or pulmonary embolism.Chronic pulmonary hypertension is characterized by major structuralchanges in the pulmonary vasculature, which result in a decreasedcross-sectional area of the pulmonary blood vessels. This may be causedby, for example, chronic hypoxia, thromboembolism, collagen vasculardiseases, pulmonary hypercirculation due to left-to-right shunt, HIVinfection, portal hypertension or a combination of genetic mutation andunknown causes as in idiopathic pulmonary arterial hypertension.

Pulmonary hypertension has been implicated in several life-threateningclinical conditions, such as adult respiratory distress syndrome(“ARDS”) and persistent pulmonary hypertension of the newborn (“PPHN”).Zapol et al., Acute Respiratory Failure, p. 241-273, Marcel Dekker, NewYork (1985); Peckham, J. Ped. 93:1005 (1978). PPHN, a disorder thatprimarily affects full-term infants, is characterized by elevatedpulmonary vascular resistance, pulmonary arterial hypertension, andright-to-left shunting of blood through the patent ductus arteriosus andforamen ovale of the newborn's heart. Mortality rates range from 12-50%.Fox, Pediatrics 59:205 (1977); Dworetz, Pediatrics 84:1 (1989).Pulmonary hypertension may also ultimately result in a potentially fatalheart condition known as “cor pulmonale,” or pulmonary heart disease.Fishman, “Pulmonary Diseases and Disorders” 2^(nd) Ed., McGraw-Hill, NewYork (1988).

Currently, there is no treatment for pulmonary hypertension that can beadministered using a compact inhalation device, such as a metered doseinhaler.

SUMMARY OF THE INVENTION

One embodiment is a method of delivering to a subject in need thereof atherapeutically effective amount of treprostinil, or treprostinilderivative or a pharmaceutically acceptable salt thereof comprisingadministering to the subject a therapeutically effective amount of thetreprostinil or treprostinil derivative or a pharmaceutically acceptablesalt thereof using a metered dose inhaler.

Another embodiment is a method for treating pulmonary hypertensioncomprising administering to a subject in need thereof treprostinil orits derivative, or a pharmaceutically acceptable salt thereof using ametered dose inhaler.

Yet another embodiment is a kit comprising a metered dose inhalercontaining a pharmaceutical formulation comprising treprostinil ortreprostinil derivative, or a pharmaceutically acceptable salt thereof.

And yet another embodiment is a kit for treating pulmonary hypertensionin a subject, comprising (i) an effective amount of treprostinil or itsderivative, or a pharmaceutically acceptable salt thereof; (ii) ametered dose inhaler; (iii) instructions for use in treating pulmonaryhypertension.

Administration of treprostinil using a metered dose inhaler can providepatients, such as pulmonary hypertension patients, with a high degree ofautonomy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 pulmonary and systemic changes in hemodynamics following theinhalation of placebo (open circles), 30 μg treprostinil (triangles), 45μg treprostinil (squares) or 60 μg TREprostinil (black circles) appliedby a Metered Dose Inhaler (MDI-TRE). A single short inhalation oftreprostinil induced sustained reduction of PAP and PVR that outlastedthe observation period of 120 minutes at doses of 45 and 60 μg MDI-TRE.Systemic arterial pressure and resistance were not significantlyaffected. PAP=mean pulmonary artery pressure; PVR=pulmonary vascularresistance; SAP=mean systemic arterial pressure; SVR=systemic vascularresistance. Data are given as mean value±standard error of the mean(SEM).

FIG. 2 presents hemodynamic changes induced by the inhalation of placebo(open circles), 30 μg treprostinil (triangles), 45 μg treprostinil(squares) or 60 μg treprostinil (black circles) applied by a metereddose inhaler. Treprostinil induced sustained elevation of cardiacoutput. Heart rate was rather unchanged as a sign for low spillover ofMDI-TRE to the systemic circulation. Gas exchange was not negativelyaffected. CO=cardiac output; HR=heart rate; SaO2=arterial oxygensaturation; SvO2=central venous oxygen saturation. Data are given asmean value±SEM.

FIG. 3 shows areas under the curve for changes in pulmonary vascularresistance (PVR) calculated for an observation period of 120 minutesafter inhalation treprostinil using a metered dose inhaler. PVR wasmarkedly lowered by treprostinil inhalation. The increased pulmonaryvasodilation over time with the two highest doses mainly relies on themore sustained effect over time. Data are shown as mean value±95%confidence intervals.

FIG. 4 demonstrates Ventilation-perfusion matching measured with themultiple inert gas elimination technique. Five patients (30 μg TRE, n=2;45 μg TRE, n=1; 60 μg TRE, n=2) with pre-existing gas exchange problemswere investigated for changes in ventilation-perfusion ratios. Allpatients had significant shunt flow at baseline. Shunt-flow and low V/Qareas were not significantly changed by nitric oxide (NO) inhalation ortreprostinil inhalation using a metered dose inhaler (MDI-TRE). MDI-TREapplied at high treprostinil concentrations did not negatively affectventilation-perfusion matching and gas-exchange. Data are given as meanvalue±95% confidence intervals.

FIG. 5 presents response of pulmonary vascular resistance (PVR) toinhaled treprostinil vs. iloprost—period effects. a) First inhalationwith treprostinil (n=22) vs. first inhalation with iloprost (n=22); b)second inhalation with treprostinil (n=22) vs. second inhalation withiloprost (n=22). The PVR decrease with treprostinil was delayed andprolonged, compared to iloprost. Due to carryover effects from the firstperiod, in the second period, the effects of both drugs appearedshortened. Data are shown as percent of baseline values (mean value±95%confidence interval).

FIG. 6 presents response of PVR and systemic arterial pressure (SAP) toinhalation of treprostinil vs. iloprost—dose effects. a) Inhalation of7.5 μg iloprost (in 6 min) vs. 7.5 μg treprostinil (6 min) (n=14, in arandomized order). b) Inhalation of 7.5 μg iloprost (6 min) vs. 15 μgtreprostinil (6 min) (n=14, in randomized order). c) Inhalation of 7.5μg iloprost (6 min) vs. 15 μg treprostinil (3 min) (n=16, in randomizedorder). Data are shown as percent of baseline values (mean±95%confidence interval). Iloprost, filled circles; Treprostinil, opentriangles.

FIG. 7 presents hemodynamic response to inhalation of treprostinil vs.iloprost. Data from n=44 patients, who inhaled both drugs in randomizedorder, shown as percent of baseline values (mean value±95% confidenceinterval). PVR, pulmonary vascular resistance; PAP, mean pulmonaryarterial pressure; SAP, mean systemic arterial pressure; CO, cardiacoutput.

FIG. 8 presents pharmacodynamics after treprostinil inhalation vs.placebo. Placebo or treprostinil in doses of 30 μg, 60 μg or 90 μg wereinhaled (means±95% confidence intervals). Maximal decrease of PVR wascomparable for all doses. The duration of pulmonary vasodilation(PVR-decrease) appeared to be dose dependent. PVR, pulmonary vascularresistance; PAP, mean pulmonary arterial pressure; SAP, mean systemicarterial pressure; CO, cardiac output; SaO2, arterial oxygen saturation;SvO2, mixed venous oxygen saturation.

FIG. 9 presents Areas Between the placebo and the treprostinil Curves(ABC). ABCs were calculated for a 3-hour period after inhalation of TREor placebo from the relative changes of hemodynamic parameters(means±95% confidence intervals). PVR, pulmonary vascular resistance;PAP, mean pulmonary arterial pressure; SAP, mean systemic arterialpressure; SVR, systemic vascular resistance.

FIG. 10 presents hemodynamic responses to the inhalation of 15 μgtreprostinil. The inhalation time by increasing treprostinilconcentration. A pulse of aerosol was generated every 6 seconds. TREaerosol was inhaled in concentrations of 100 μg/ml (18 pulses; n=6), 200μg/ml (9 pulses; n=6), 600 μg/ml (3 pulses; n=21), 1000 μg/ml (2 pulses;n=7) and 2000 μg/ml (1 pulse; n=8). Placebo data correspond to FIG. 8.Data are shown as means±95% confidence intervals. PVR, pulmonaryvascular resistance; PAP, mean pulmonary arterial pressure; SAP, meansystemic arterial pressure; CO, cardiac output.

FIG. 11 presents areas between the placebo curve and the responses to 15μg treprostinil applied at increasing concentrations to minimizeinhalation time. Mean±SEM of relative changes of hemodynamic parameters(observation time 120 min). PAP, pulmonary arterial pressure, SAP,systemic arterial pressure, PVR, pulmonary vascular resistance, CO,cardiac output, SaO2, systemic arterial oxygen saturation, SvO2,pulmonary arterial oxygen saturation.

FIG. 12 presents pharmacokinetics of treprostinil after a singleinhalation. Treprostinil plasma levels after inhalation of 30 μg, 60 μg,90 μg or 120 μg treprostinil (6 min inhalation period; experimentscorrespond to those shown in FIGS. 8 and 9). Data with error barsrepresent mean values±SEM.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise specified, the term “a” or “an” used herein shall mean“one or more.”

The present application incorporates herein by reference in its entiretyVoswinckel R, et al. J. Am. Coll. Cardiol. 2006; 48:1672-1681.

The inventors discovered that a therapeutically effective dose oftreprostinil can be administered in a few single inhalations using acompact inhalation device, such as a metered dose inhaler. Furthermore,the inventors discovered that such administering does not causesignificant side effects, especially no significant side effects relatedto systemic blood pressure and circulation as well as no gas exchangedeteriorations or disruptions.

Accordingly, one embodiment of the invention is a method of deliveringto a subject in need thereof, such as a human being, a therapeuticallyeffective amount of treprostinil comprising administering to the subjecta formulation comprising a therapeutically effective amount oftreprostinil, its derivative or a pharmaceutically acceptable saltthereof using a metered dose inhaler. Treprostinil can be administeredvia a metered dose inhaler to a subject affected with a condition ordisease, which can be treated by treprostinil, such as asthma, pulmonaryhypertension, peripheral vascular disease or pulmonary fibrosis.

Another embodiment of the invention is a method for treating pulmonaryhypertension, comprising administering to a subject in need thereof,such as a human being, treprostinil or its derivative, or apharmaceutically acceptable salt using a metered dose inhaler.

Treprostinil, or9-deoxy-2′,9-alpha-methano-3-oxa-4,5,6-trinor-3,7-(1′3′-interphenylene)-13,14-dihydro-prostaglandinF1, is a prostacyclin analogue, first described in U.S. Pat. No.4,306,075. U.S. Pat. No. 5,153,222 describes use of treprostinil fortreatment of pulmonary hypertension. Treprostinil is approved for theintravenous as well as subcutaneous route, the latter avoiding septicevents associated with continuous intravenous catheters. U.S. Pat. Nos.6,521,212 and 6,756,033 describe administration of treprostinil byinhalation for treatment of pulmonary hypertension, peripheral vasculardisease and other diseases and conditions. U.S. Pat. No. 6,803,386discloses administration of treprostinil for treating cancer such aslung, liver, brain, pancreatic, kidney, prostate, breast, colon andhead-neck cancer. US patent application publication No. 2005/0165111discloses treprostinil treatment of ischemic lesions. U.S. Pat. No.7,199,157 discloses that treprostinil treatment improves kidneyfunctions. US patent application publication No. 2005/0282903 disclosestreprostinil treatment of neuropathic foot ulcers. U.S. provisionalapplication No. 60/900,320 filed Feb. 9, 2007, discloses treprostiniltreatment of pulmonary fibrosis.

The term “acid derivative” is used herein to describe C1-4 alkyl estersand amides, including amides wherein the nitrogen is optionallysubstituted by one or two C1-4 alkyl groups.

The present invention also encompasses methods of using Treprostinil orits derivatives, or pharmaceutically acceptable salts thereof. In oneembodiment, a method uses Treprostinil sodium, currently marketed underthe trade name of REMODULIN®. The FDA has approved Treprostinil sodiumfor the treatment of pulmonary arterial hypertension by injection ofdose concentrations of 1.0 mg/mL, 2.5 mg/mL, 5.0 mg/mL and 10.0 mg/mL.The chemical structure formula for Treprostinil sodium is:

Treprostinil sodium is sometimes designated by the chemical names: (a)[(1R,2R,3aS,9aS)-2,3,3a,4,9,9a-hexahydro-2-hydroxy-1-[(3S)-3-hydroxyoctyl]-1H-benz[f]inden-5-yl]oxy]aceticacid; or (b)9-deoxy-2′,9-α-methano-3-oxa-4,5,6-trinor-3,7-(1′,3′-interphenylene)-13,14-dihydro-prostaglandinF₁. Treprostinil sodium is also known as: UT-15; LRX-15; 15AU81;UNIPROST™; BW A15AU; and U-62,840. The molecular weight of Treprostinilsodium is 390.52, and its empirical formula is C₂₃H₃₄O₅.

In certain embodiments, treprostinil can be administered in combinationwith one or more additional active agents. In some embodiments, such oneor more additional active agents can be also administered together withtreprostinil using a metered dose inhaler. Yet in some embodiments, suchone or more additional active agents can be administered separately fromtreprostinil. Particular additional active agents that can beadministered in combination with treprostinil may depend on a particulardisease or condition for treatment or prevention of which treprostinilis administered. In some cases, the additional active agent can be acardiovascular agent such as a calcium channel blocker, aphosphodiesterase inhibitor, an endothelial antagonist, or anantiplatelet agent.

The present invention extends to methods of using physiologicallyacceptable salts of Treprostinil, as well as non-physiologicallyacceptable salts of Treprostinil that may be used in the preparation ofthe pharmacologically active compounds of the invention.

The term “pharmaceutically acceptable salt” refers to a salt ofTreprostinil with an inorganic base, organic base, inorganic acid,organic acid, or basic or acidic amino acid. Salts of inorganic basescan be, for example, salts of alkali metals such as sodium or potassium;alkaline earth metals such as calcium and magnesium or aluminum; andammonia. Salts of organic bases can be, for example, saltstrimethylamine, triethylamine, pyridine, picoline, ethanolamine,diethanolamine, and triethanolamine. Salts of inorganic acids can be,for example, salts of hydrochloric acid, hydroboric acid, nitric acid,sulfuric acid, and phosphoric acid. Salts of organic acids can be, forexample, salts of formic acid, acetic acid, trifluoroacetic acid,fumaric acid, oxalic acid, lactic acid, tartaric acid, maleic acid,citric acid, succinic acid, malic acid, methanesulfonic acid,benzenesulfonic acid, and p-toluenesulfonic acid. Salts of basic aminoacids can be, for example, salts of arginine, lysine and ornithine.Salts of acidic amino acids can include, for example, salts of asparticacid and glutamic acid. Quaternary ammonium salts can be formed, forexample, by reaction with lower alkyl halides, such as methyl, ethyl,propyl, and butyl chlorides, bromides, and iodides, with dialkylsulphates, with long chain halides, such as decyl, lauryl, myristyl, andstearyl chlorides, bromides, and iodides, and with aralkyl halides, suchas benzyl and phenethyl bromides.

Preferred pharmaceutically acceptable salts are disclosed, for example,in U.S. patent application publication No. 20050085540.

Treprostinil can be administered by inhalation, which in the presentcontext refers to the delivery of the active ingredient or a combinationof active ingredients through a respiratory passage, wherein the subjectin need of the active ingredient(s) through the subject's airways, suchas the subject's nose or mouth.

A metered dose inhaler in the present context means a device capable ofdelivering a metered or bolus dose of respiratory drug, such astreprostinil, to the lungs. One example of the inhalation device can bea pressurized metered dose inhaler, a device which produces the aerosolclouds for inhalation from solutions and/or suspensions of respiratorydrugs in chlorofluorocarbon (CFC) and/or hydrofluroalkane (HFA)solutions.

The inhalation device can be also a dry powder inhaler. In such case,the respiratory drug is inhaled in solid formulation, usually in theform of a powder with particle size less than 10 micrometers in diameteror less than 5 micrometers in diameter.

The metered dose inhaler can be a soft mist inhaler (SMI), in which theaerosol cloud containing a respiratory drug can be generated by passinga solution containing the respiratory drug through a nozzle or series ofnozzles. The aerosol generation can be achieved in SMI, for example, bymechanical, electromechanical or thermomechanical process. Examples ofsoft mist inhalers include the Respimat® Inhaler (Boeringer IngelheimGmbH), the AERx® Inhaler (Aradigm Corp.), the Mystic™ Inhaler (VentairaPharmaceuticals, Inc) and the Aira™ Inhaler (Chrysalis TechnologiesIncorporated). For a review of soft mist inhaler technology, see e.g. M.Hindle, The Drug Delivery Companies Report, Autumn/Winter 2004, pp.31-34. The aerosol for SMI can be generated from a solution of therespiratory drug further containing pharmaceutically acceptableexcipients. In the present case, the respiratory drug is treprostinil,its derivative or a pharmaceutically acceptable salt thereof, which canbe formulated in SMI is as a solution. The solution can be, for example,a solution of treprostinil in water, ethanol or a mixture thereof.Preferably, the diameter of the treprostinil-containing aerosolparticles is less than about 10 microns, or less than about 5 microns,or less than about 4 microns.

Treprostinil concentration in an aerosolable formulation, such as asolution, used in a metered dose inhaler can range from about 500 μg/mlto about 2500 μg/ml, or from about 800 μg/ml to about 2200 μg/ml, orfrom about 1000 μg/ml to about 2000 μg/ml.

The dose of treprostinil that can be administered using a metered doseinhaler in a single event can be from about 15 μg to about 100 μg orfrom about 15 μg to about 90 μg or from about 30 μg to about 90 μg orfrom about 30 μg to about 60 μg.

Administering of treprostinil in a single event can be carried out in alimited number of breaths by a patient. For example, treprostinil can beadministered in 20 breaths or less, or in 10 breaths or less, or than 5breaths or less. Preferably, treprostinil is administered in 3, 2 or 1breaths.

The total time of a single administering event can be less than 5minutes, or less than 1 minute, or less than 30 seconds.

Treprostinil can be administered a single time per day or several timesper day.

In some embodiments, the method of treatment of pulmonary hypertensioncan further comprise administering at least one supplementary agentselected from the group consisting of sildenafil, tadalafil, calciumchannel blockers (diltiazem, amlodipine, nifedipine), bosentan,sitaxsentan, ambrisentan, and pharmaceutically acceptable salts thereof.In some embodiments, the supplementary agents can be included in thetreprostinil formulation and, thus, can be administered simultaneouslywith treprostinil using a metered dose inhaler. In some embodiments, thesupplementary agents can be administered separately from treprostinil.In some embodiments, the application of intravenous prostacyclin(flolan), intravenous iloprost or intravenous or subcutaneoustreprostinil can be administered in addition to treprostiniladministered via inhalation using a metered dose inhaler.

The present invention also provides a kit that includes a metered doseinhaler containing a pharmaceutical formulation comprising treprostinilor its derivative, or a pharmaceutically acceptable salt thereof. Such akit can further include instructions on how to use the metered doseinhaler for inhaling treprostinil. Such instructions can include, forexample, information on how to coordinate patient's breathing, andactuation of the inhaler. The kit can be used by a subject, such ashuman being, affected with a disease or condition that can be treated bytreprostinil, such as asthma, pulmonary hypertension, peripheralvascular disease or pulmonary fibrosis.

In some cases, the kit is a kit for treating pulmonary hypertension,that includes (i) a metered dose inhaler containing a pharmaceuticalformulation comprising treprostinil or its derivative, or apharmaceutically acceptable salt thereof; and (ii) instructions for useof the metered dose inhaler containing treprostinil in treatingpulmonary hypertension.

As used herein, the phrase “instructions for use” shall mean anyFDA-mandated labeling, instructions, or package inserts that relate tothe administration of Treprostinil or its derivatives, orpharmaceutically acceptable salts thereof, for treatment of pulmonaryhypertension by inhalation. For example, instructions for use mayinclude, but are not limited to, indications for pulmonary hypertension,identification of specific symptoms associated with pulmonaryhypertension, that can be ameliorated by Treprostinil, recommendeddosage amounts for subjects suffering from pulmonary hypertension andinstructions on coordination of individual's breathing and actuation ofthe metered dose inhaler.

The present invention can be illustrated in more detail by the followingexample, however, it should be understood that the present invention isnot limited thereto.

EXAMPLE 1 Open Label Study Upon Acute Safety, Tolerability andHemodynamic Effects of Inhaled Treprostinil Delivered in Seconds

A study was conducted of acute vasodilator challenge during right heartcatheter investigation to determine the safety, tolerability andpulmonary vasodilatory potency of inhaled treprostinil applied inseconds by a soft mist inhaler (SMI-TRE). The study produced evidencefor a long lasting favourable effect of SMI-TRE on pulmonaryhemodynamics in absence of systemic side effects and gas exchangedisruptions.

Summary

Inhaled nitric oxide (20 ppm; n=45) and inhaled treprostinil sodium(TRE; n=41) or placebo (n=4) were applied once during right heartcatheter investigation. TRE was delivered in 2 breaths (1000 μg/mlaerosol concentration; 30 μg dose; n=12), 3 breaths (1000 μg/ml; 45 μg;n=9) or 2 breaths (2000 μg/ml; 60 μg; n=20) from a Respimat® SMI.Pulmonary hemodynamics and blood gases were measured at defined timepoints, observation time following TRE application was 120 minutes. TREdoses of 30 μg, 45 μg and 60 μg reduced pulmonary vascular resistance(PVR) to 84.4±8.7%, 71.4±17.5% and 77.5±7.2% of baseline values,respectively (mean±95% confidence interval). The 120 minute area underthe curve for PVR for placebo, 30 μg, 45 μg and 60 μg TRE was 1230±1310,−870±940, −2450±2070 and −2000±900 min %, respectively. Reduction of PVRby a single inhalation of the two higher doses outlasted the observationperiod of 120 minutes. Reduction of systemic vascular resistance andpressure was negligible, showing a high pulmonary selectivity forSMI-TRE. Intrapulmonary selectivity was also provided by SMI-TRE asventilation/perfusion matching, assessed by the multiple inert gaselimination technique in 5 patients with gas exchange problems, was notsignificantly different after SMI-TRE compared to inhaled nitric oxideor no treatment. No significant side effects were observed.

Conclusions: The acute application of inhaled treprostinil with ametered dose inhaler in 2-3 breaths was safe, well tolerated and induceda strong and sustained pulmonary selective vasodilation.

Methods and Patients

A total number of 45 patients with moderate to severe precapillarypulmonary hypertension were enrolled. Patient characteristics were:female to male ratio (f/m)=29/16, age 59±2.3 years, pulmonary arterypressure (PAP) 45±1.8 mmHg, pulmonary vascular resistance (PVR) 743±52dynes·s·cm⁻⁵, pulmonary artery wedge pressure (PAWP) 8.6±0.5 mmHg,central venous pressure (CVP) 6.4±0.7 mmHg, cardiac output (CO) 4.5±0.2l/min, central venous oxygen saturation (SvO2) 62.3±1.2 mmHg(mean±Standard Error of the Mean). Disease etiologies were idiopathicPAH (iPAH) (n=13), PAH other (n=11), chronic thromboembolic pulmonaryhypertension (CTEPH) (n=17) and pulmonary fibrosis (n=4). Table 1presents the patient characteristics of the different groups.

TABLE 1 Patient characteristics of the different treatment groups.Placebo 30 μg TRE 45 μg TRE 60 μg TRE (n = 4) (n = 12) (n = 9) (n = 20)Age [years] 61 ± 8 53.9 ± 3.9 54.2 ± 5.7 65.5 ± 3.1 PAP [mmHg]  49.5 ±10.1   45 ± 3.1 54.3 ± 2.8 39.7 ± 2.0 PVR [Dynes]  896 ± 163   597 ±53.9 1049 ± 107 663 ± 81 CO [l/min] 4.46 ± 0.9  5.2 ± 0.4  3.9 ± 0.4 4.4 ± 0.3 SAP [mmHg]   98 ± 8.1 90.1 ± 3.2 82.8 ± 3.9 86.1 ± 2.0 SaO2[%] 85.3 ± 4.5 90.0 ± 1.1 89.6 ± 1.1 90.6 ± 0.5 SvO2 [%] 57.5 ± 3.9 66.0± 1.6 59.1 ± 3.4 62.5 ± 1.6 Data are given as mean ± Standard Error ofthe Mean (SEM). PAP = pulmonary artery pressure; PVR = pulmonaryvascular resistance; CO = cardiac output; SAP = systemic arterialpressure; SaO2 = arterial oxygen saturation; SvO2 = central venousoxygen saturation.

Baseline values were determined 20-30 minutes after placement of thecatheter. Heart rate, pulmonary and systemic blood pressure and cardiacoutput were measured and blood gases were taken during eachpharmacological intervention at defined time points. Pharmacologicalinterventions included the inhalation of 20 ppm nitric oxide (NO) afterevaluation of baseline parameters (n=45) and the consecutive inhalationof placebo (n=4), 30 μg SMI-TRE (n=12), 45 μg SMI-TRE (n=9) or 60 μg(n=20) SMI-TRE. Placebo and treprostinil was applied with the Respimat®SMI. For filling of this device with treprostinil sodium, the placebosolution was withdrawn from the device with a syringe and treprostinilsolution was injected into the device under sterile conditions. Aerosolquality was controlled before and after refilling of the SMI devices bylaser diffractometry, see e.g. Gessler T., Schmehl T., Hoeper M. M.,Rose F., Ghofrani H. A., Olschewski H. et al. Ultrasonic versus jetnebulization of iloprost in severe pulmonary hypertension. Eur. Respir.J. 2001; 17:14-19 incorporated herein in its entirety. The aerosol sizesbefore (placebo) and after filling (treprostinil) were unchanged. Theaerosol particles mass median aerodynamic diameter oftreprostinil-aerosol was 4-5 μm, which can be at the upper limit foralveolar deposition. The aerosol volume delivered by one cycle from theSMI was 15 μl. The solution used for aerosol generation was preparedfrom treprostinil sodium salt using a standard protocol. The SMI waseither filled with a concentration of 1000 μg/ml treprostinil sodium(one aerosol puff=15 μg TRE) or with 2000 μg/ml (one puff=30 μg TRE).The different doses were applied as 2 puffs 1000 μg/ml (30 μg), 3 puffs1000 μg/ml (45 μg) and 2 puffs 2000 μg/ml (60 μg). The placebo wasinhaled as 2 puffs from a placebo-SMI. Hemodynamics and gas-exchangeparameters were recorded for 120 minutes after TRE inhalation. Thisstudy used the Respimat® device, because the implemented “soft mist”technology was well suited for the deposition of such highly activedrugs like prostanoids.

The impact of SMI-TRE on ventilation-perfusion matching was assessed infive patients (30 μg TRE, n=2; 45 μg TRE, n=1; 60 μg TRE, n=2) withpre-existing gas exchange problems by use of the multiple inert gaselimination technique (MIGET), see e.g. Wagner P D, Saltzman H A, West JB. Measurement of continuous distributions of ventilation-perfusionratios: theory. J Appl Physiol. 1974; 36:588-99; Ghofrani H A, WiedemannR, Rose F, Schermuly R T, Olschewski H, Weissmann N et al. Sildenafilfor treatment of lung fibrosis and pulmonary hypertension: a randomisedcontrolled trial. Lancet. 2002; 360:895-900, both incorporated herein intheir entirety.

Statistics:

Mean values, standard deviation, standard error of the mean and 95%confidence intervals were calculated. Statistical analysis was done byuse of a paired t-test.

Results:

The inhalation of treprostinil sodium from the metered dose inhaler(SMI-TRE) was well tolerated, only mild and transient cough for amaximum of one minute was reported. No systemic side effects likeheadache, flush, nausea or dizziness were observed.

Two to three breaths of SMI-TRE induced a strong pulmonary vasodilationthat outlasted the observation time of 120 minutes (45 and 60 μg). Thelower dose of 30 μg TRE induced a somewhat shorter effect on pulmonaryvascular resistance; however, the maximal pulmonary vasodilation wascomparable. In contrast, placebo inhalation did not induce pulmonaryvasodilation. In fact a slight increase in PVR over the time of theright heart catheter investigation could be recorded following placeboinhalation (FIG. 1). The effect of SMI-TRE on systemic vascularresistance and pressure was very small and not clinically significant.Cardiac output was significantly increased over the whole observationperiod, whereas heart rate was rather unchanged. Gas exchange was notinfluenced by SMI-TRE (FIG. 2). The maximal changes in hemodynamic andgas-exchange parameters compared to baseline values are depicted inTable 2.

TABLE 2 Extremes of the relative changes of hemodynamic and gas exchangeparameters compared to baseline after inhalation of Placebo (n = 4), 30μg treprostinil (n = 12), 45 μg treprostinil (n = 9) and 60 μgtreprostinil (n = 20). Placebo 30 μg TRE 45 μg TRE 60 μg TRE PAP (min) 99.4 ± 3.0 83.4 ± 3.2 77.6 ± 6.8 79.5 ± 2.4 PVR (min) 101.4 ± 1.9 84.4± 4.4 71.4 ± 8.9 77.5 ± 3.7 CO (max)  99.7 ± 1.1 108.8 ± 3.8  108.6 ±5.6  103.8 ± 2.0  SVR (min) 104.3 ± 4.3 97.7 ± 4.2   92 ± 3.9 91.3 ± 2.1SAP (min) 102.7 ± 1.7 97.3 ± 1.9 96.1 ± 1.5 93.6 ± 2.9 HR (max)   105 ±2.1 106.1 ± 2.9  99.1 ± 2.4 101.1 ± 0.9  SaO2 (min)  98.2 ± 0.4  101 ±0.3 94.4 ± 1.8 95.8 ± 0.9 SvO2 (max) 104.5 ± 1.4 102.4 ± 1.3  104.5 ±4.4   102 ± 1.0 Highest (max) and lowest (min) values during theobservation period are shown. Data are given as percent of baselinevalues (mean ± SEM). PAP = pulmonary artery pressure; PVR = pulmonaryvascular resistance; SVR = systemic vascular resistance; CO = cardiacoutput; SAP = systemic arterial pressure; HR = heart rate; SaO2 =arterial oxygen saturation; SvO2 = central venous oxygen saturation.

The areas under the curve for PVR were calculated for placebo and thedifferent SMI-TRE doses over the 120 minute observation period (FIG. 3).A dose effect of SMI-TRE with a trend to a more sustained effect withthe two highest doses could be observed.

The inhalation of a highly concentrated aerosol can be in theory proneto disturbances of gas exchange because the deposition of even smallamounts of aerosol may deliver high doses locally and thereby antagonizethe hypoxic pulmonary vasoconstriction in poorly ventilated areas. Thiswould then lead to increased shunt flow or increase of lowventilation/perfusion (V/Q) areas. This question was addressed in fivepatients with the multiple inert gas elimination technique (MIGET), thegold-standard for intrapulmonary V/Q ratio determination. The MIGETpatients were selected for pre-existing gas exchange limitations.Characteristics of these patients were: PAP 54.6±3.2 mmHg, PVR 892±88dynes, SaO2 91.7±0.5%, SvO2 65.2±1.8%. Etiologies were iPAH (n=1), CTEPH(n=3), pulmonary fibrosis (n=1). The maximal relative reduction of SaO2after inhalation of SMI-TRE in these patients was −3.8±1.5% compared tobaseline values. Shunt flow at baseline, NO-inhalation and 60 minutesafter SMI-TRE was 6.4±4.3%, 5.4±3.0% and 8.3±3.4%, respectively(mean±95% confidence interval; FIG. 4).

No significant increase in low V/Q areas or shunt fraction afterinhalation of SMI-TRE was observed, in fact the distribution ofperfusion was not different to that at baseline and during nitric oxideinhalation. This proves an excellent intrapulmonary selectivity ofSMI-TRE, which is also reflected by unchanged arterial oxygensaturation.

Conclusion:

Treprostinil is tolerated at high doses with no systemic side effects.The application of an effective amount of treprostinil in only few oreven one single breath was achieved with a highly concentratedtreprostinil sodium solution. Treprostinil can be applied by a metereddose inhaler, such as Respimat® soft mist inhaler.

EXAMPLE 2 Investigation of The Effects of Inhaled Treprostinil onPulmonary Hemodynamics and Gas Exchange in Severe Pulmonary Hypertension

This study investigated the effects of inhaled treprostinil on pulmonaryvascular resistance in severe pulmonary hypertension and addressedsystemic effects and gas exchange as well as tolerability and efficacyof high doses of treprostinil given in short time. A total of 123patients with a mean pulmonary artery pressure of about 50 mmHg wereinvestigated in three separate randomized studies. Inhaled treprostinilexerted potent sustained pulmonary vasodilation with excellenttolerability and could be safely applied in a few breaths or even onebreath.

Summary:

Three different studies were conducted on a total of 123 patients bymeans of right heart catheterization: i) a randomized crossover-designstudy (44 patients), ii) a dose escalation study (31 patients) and iii)a study of reduction of inhalation time while keeping the dose fixed (48patients). The primary endpoint was the change in pulmonary vascularresistance (PVR).

The mean pulmonary artery pressure of the enrolled patients was about 50mmHg. Hemodynamics and patient characteristics were similar in allstudies. In study i) TRE and Iloprost (ILO), at an inhaled dose of 7.5μg, displayed comparable PVR decrease, with a significantly differenttime course (p<0.001), TRE exhibiting a more sustained effect on PVR(p<0.0001) and less systemic side effects. In study ii) placebo, 30 μg,60 μg, 90 μg or 120 μg TRE were applied with drug effects being observedfor 3 hours after inhalation. A near-maximal acute PVR decrease wasobserved at 30 μg TRE. In study iii) TRE was inhaled with a pulsedultrasonic nebulizer, mimicking a metered dose inhaler. 15 μg TRE wasinhaled with 18 pulses (TRE concentration 100 μg/ml), 9 pulses (200μg/ml), 3 pulses (600 μg/ml), 2 pulses (1000 μg/ml) or 1 pulse (2000μg/ml), each mode achieving comparable, sustained pulmonaryvasodilation.

Inhaled treprostinil exerts sustained pulmonary vasodilation withexcellent tolerability at doses, which may be inhaled in a few or evenone breath. Inhaled treprostinil is advantageous to inhaled iloprost interms of duration of effect and systemic side effects. Inhaledtreprostinil is well tolerated in concentrations up to 2000 mg/ml(bringing down inhalation time to a single breath) and in high doses (upto 90 μg).

Methods:

All inhalations were performed with the Optineb® ultrasonic nebulizer(Nebutec, Elsenfeld, Germany).

Study i) was a randomized, open-label, single-blind crossover study. Theprimary objective was to compare the acute hemodynamic effects and thesystemic side effects of inhaled treprostinil with inhaled iloprost atcomparable doses. A total number of 44 patients with moderate to severeprecapillary pulmonary hypertension were enrolled. Patientcharacteristics and hemodynamic as well as gas exchange parameters areoutlined in Table 3.

TABLE 3 Patient characteristics, hemodynamic parameters and gas exchangevalues at baseline, before challenge with inhalative prostanoids. GenderEtiology PAP PVR SAP CVP PAWP CO SaO2 SvO2 N Age f/m i/o/t/f [mmHg][dyn*s*cm⁻⁵] [mmHg] [mmHg] [mmHg] [l/min] [%] [%] 1a 14 55.1 ± 4.8 11/3 4/4/2/4 53.8 ± 3.1 911 ± 102 95.4 ± 3.6  7.4 ± 1  8.0 ± 0.8 4.3 ± 0.493.8 ± 2 63.9 ± 2.4 1b 14 54.1 ± 3.3 10/4  1/6/5/2 47.4 ± 3.8 716 ± 80 90.6 ± 3.3  5.9 ± 1.4  6.4 ± 0.7 4.7 ± 0.4   92 ± 1 64.4 ± 2.3 1c 16  56 ± 2.9 7/9 6/3/6/1 47.5 ± 4.5 777 ± 102   92 ± 4.5  8.3 ± 1.4  8.6 ±1.4 4.4 ± 0.5 91.4 ± 0.9 59.8 ± 2.6 2a 8 60.8 ± 4 4/4 2/2/3/1 51.9 ± 4.9849 ± 152 95.9 ± 4.8  7.6 ± 1.4 11.1 ± 1.7 4.4 ± 0.6 89.6 ± 2.8 60.1 ±2.8 2b 8 52.8 ± 6.6 6/2 1/3/3/1   49 ± 4 902 ± 189 92.4 ± 2.4  4.8 ± 1.1 7.2 ± 1.3 4.0 ± 0.4 92.4 ± 2.4 62.5 ± 1.7 2c 6 56.8 ± 5.9 4/2 0/2/2/244.2 ± 3.5 856 ± 123 96.3 ± 3.9   5 ± 1.1   6 ± 1 3.8 ± 0.3 92.8 ± 1.563.6 ± 1.8 2d 6 51.2 ± 3.8 4/2 2/2/2/0 55.5 ± 4.9 940 ± 110 91.2 ± 8.111.2 ± 1.2   10 ± 0.7 3.9 ± 0.4   92 ± 1.9   62 ± 5.8 2e 3 57.3 ± 9.11/2 0/1/0/2 45.3 ± 5.2 769 ± 267   99 ± 3.2   5 ± 2.1   9 ± 0.6 4.5 ±0.6 94.2 ± 1.3 66.3 ± 1.5 3a 6 52.7 ± 6.6 4/2 2/4/0/0 53.8 ± 6.7 928 ±145 92.7 ± 7.9  8.7 ± 2.7  8.8 ± 1.3 4.2 ± 0.6 90.4 ± 2.8 64.8 ± 4.3 3b6 58.3 ± 3.5 4/2 3/1/1/1 54.2 ± 6.1 808 ± 156 94.3 ± 2.8   7 ± 1.4  10 ±1.3   5 ± 0.7 91.9 ± 0.7 63.5 ± 2.9 3c 21 57.4 ± 5.6 8/3 7/7/6/1 46.1 ±2.5 900 ± 99    88 ± 2.8   9 ± 1.4  9.2 ± 0.5 3.7 ± 0.3 91.7 ± 0.5 59.7± 2 3d 7 55.6 ± 5.8 3/4 0/4/3/0 53.1 ± 7.1 732 ± 123 91.4 ± 5.6  7.9 ±3.1  8.6 ± 1.3   5 ± 0.4 90.7 ± 1.4 61.3 ± 3.7 3e 8   59 ± 5.2 7/10/4/4/0 45.1 ± 3.9 733 ± 114 92.8 ± 6.8  4.6 ± 0.8  8.1 ± 1.1 4.3 ± 0.290.7 ± 0.8 66.3 ± 2.8 Group 1 corresponds to study i); randomizedcrossover study comparing inhaled iloprost (ILO) and inhaledtreprostinil (TRE). a = 7.5 g ILO vs. 7.5 μg TRE, b = 7.5 g ILO vs. 15μg TRE (6 min inhalation time), c = 7.5 g ILO vs. 15 μg TRE (3 mininhalation time). Group 2 corresponds to study ii); evaluation ofmaximal tolerated dose of TRE. a = placebo inhalation, b = 30 μg TRE, c= 60 μg TRE, d = 90 μg TRE, e = 120 μg TRE. Group 3 corresponds to studyiii); reduction of inhalation time by increase of TRE concentration,aiming at a total inhaled dose of 15 μg. a = 18 pulses of 100 μg/ml TRE,b = 9 pulses of 200 μg/ml TRE, c = 3 pulses of 600 μg/ml TRE, d = 2pulses of 1000 μg/ml TRE, e = 1 pulse 2000 μg/ml TRE. Etiology ofpulmonary hypertension was classified as idiopathic PAH (i), PAH ofother causes (o), chronic thromboembolic PH (t), and pulmonary fibrosis(f).

Each patient inhaled both iloprost and treprostinil on the same dayduring right heart catheter investigation; the drugs were administeredconsecutively with a one hour interval between the drug applications.One half of the study patients initially inhaled treprostinil and theninhaled iloprost (n=22), while the other half initially inhaled iloprostand then inhaled treprostinil (n=22). Patients were randomized to one ofthe two groups and blinded as to the study drugs. Drug effects weremonitored for 60 minutes after each inhalation. Iloprost was inhaled at4 μg/ml (6 min inhalation time; n=44) and treprostinil was inhaled at aconcentration of 4 μg/ml (6 min inhalation; n=14), 8 μg/ml (6 mininhalation; n=14) or 16 μg/ml (3 min inhalation; n=16). Based onprevious biophysical characterization of the ultrasonic device withiloprost- and treprostinil-solution, this corresponds to a total inhaleddose of 7.5 μg iloprost and treprostinil (4 μg/ml) and 15 μgtreprostinil (8 μg/ml and 16 μg/ml), respectively.

Study ii) was a randomized, open-label, single blind, placebo controlledstudy. The primary objectives were to describe the pharmacodynamic andpharmacokinetic effects of inhaled treprostinil at a well tolerated dose(30 μg) and to explore the highest tolerated single dose. A total numberof 31 patients inhaled either placebo or treprostinil; each patientreceived one inhalation. The first 16 patients were randomized to 30 μgTRE (16 μg/ml, n=8) or placebo (stock solution in a concentrationcorresponding to TRE 16 μg/ml). Subsequent patients received 60 μg TRE(32 μg/ml; n=6), 90 μg TRE (48 μg/ml; n=6) and 120 μg TRE (64 μg/ml;n=3). Inhalation time was 6 minutes in all groups. Hemodynamics andgas-exchange as well as arterial treprostinil concentrations wererecorded for 180 minutes.

Study iii) was a randomized, open-label, single blind study. The primaryobjective was to explore the shortest possible inhalation time for a 15μg dose of inhaled treprostinil. A total of 48patients inhaled one doseof TRE during right heart catheter investigation. The drug was appliedin 18, 9, 3, 2 or 1 breaths. The aerosol was generated by a pulsedultrasonic nebulizer (VENTA-NEB®, Nebutec, Elsenfeld, Germany) in cyclesconsisting of 2seconds aerosol production (pulse) and 4seconds pause.The device included an opto-acoustical trigger for the patient tosynchronize the inspiration to the end of the aerosol pulse, therebyproviding exact dosage. The TRE dose of 15 μg was either generatedduring 18 cycles Optineb filled with 100 μg/ml TRE, n=6), 9 cycles (200μg/ml TRE, n=6), 3 cycles (600 μg/ml TRE, n=21), 2 cycles (1000 μg/mlTRE, n=7) or 1 cycle (2000 μg/ ml TRE, n=8). Hemodynamics and gasexchange were recorded for 120-180 minutes.

Treprostinil plasma concentrations were assessed in study ii) at 10, 15,30, 60 and 120 minutes after inhalation. Treprostinil quantification wasdone by Alta Analytical Laboratory (El Dorado Hills, Calif., USA) with avalidated liquid chromatography atmospheric-pressure ionization tandemmass spectrometry as previously described Wade M., et al. J. Clin.Pharmacol. 2004; 44:503-9. Mixed venous blood was drawn at the depictedtime points (FIG. 11) after inhalation, centrifuged and the plasmafrozen at −80° C. until temperature controlled shipping on dry ice.

Statistics:

For statistical analysis of study i) the repeated PVR measurements afterinhaled iloprost and treprostinil were subjected to a three-factorialanalysis of variance (ANOVA; factors: time (A), drug (B), treprostinilconcentration (C)) to avoid multiple testing. The time to maximum PVRdecrease after inhalation of iloprost versus treprostinil was comparedby paired t-test. Area under the curve (AUC) was calculated from startof inhalation until 60 min after inhalation. Means, standard error ofthe mean (SEM) and 95% confidence intervals were calculated. For studyii) and iii) areas between curves (ABC) were calculated between placeboinhalation (study ii) and the respective treprostinil inhalation until180 min (study ii)) and 120 min (study iii)) after end of inhalation.

Results:

The inhalation of iloprost as well as treprostinil in study i) resultedin a rapid decrease in PVR and PAP (FIG. 5-7). No significantdifferences were observed for the areas under the curve (AUC) of PVRdecrease after inhalation of 7.5 μg TRE in 6 minutes (AUC −12.6±7.0%),15 μg TRE in 6 minutes (AUC −13.3±3.2%) and 15 μg TRE in 3 minutes (AUC−13.6±4.3%). The AUC for PVR after the inhalation of 7.5 μg iloprost in6 minutes was −7.7±3.7% (mean±95% confidence interval). An overview ofthe pooled data of treprostinil inhalation as compared to iloprostinhalation is given in FIG. 7. The maximum effect of iloprost andtreprostinil on PVR was comparable but this effect was reachedsignificantly later after treprostinil inhalation (18±2 min) compared toiloprost (8±1 min; mean±SEM, p<0.0001) and lasted considerably longer(after 60 min, PVR values in the treprostinil group had not yet returnedto baseline). The increase in cardiac output was less acute butprolonged after treprostinil inhalation. Systemic arterial pressure(SAP) was unaffected by treprostinil inhalation, whereas a transientdecrease was observed after iloprost inhalation. Iloprost andtreprostinil did not affect gas exchange. Three-factorial ANOVA for PVRdemonstrated a significant difference between repeated measurementsafter inhalation (p_((A))<0.0001), no significant difference betweendrugs (p_(B)=0.1), no difference between treprostinil concentrations(p_((C))=0.74) and a significant drug x time interaction(p_((A×B))<0.0001). This translates into a significant effect of bothdrugs on PVR with comparable drug potency but a prolonged drug effect oftreprostinil compared to iloprost.

In this study the occasionally observed mild side effects of iloprostinhalation at the given dose (transient flush, headache) were notobserved with inhaled treprostinil. Bad taste was reported by most ofthe patients after inhalation of TRE. This was later found to beattributable to the metacresol preservative contained in thetreprostinil solution.

In study ii) pharmacodynamics of inhaled placebo or treprostinil wereobserved for 180 minutes. Placebo inhalation was followed by a gradualincrease in PVR over the entire observation time. Due to reduced patientnumbers in the 120 μg TRE group (because of side effects, see below),the hemodynamic values for this group were not included in the graphs ofthis study (FIG. 8-9). All TRE doses lead to comparable maximaldecreases of PVR to 76.5±4.7% (30 μg), 73.7±5.8% (60 μg), 73.3±4.3% (90μg) and 65.4±4.1% (120 μg) of baseline values. An extended duration ofpulmonary vasodilation was noted, surpassing the 3 hour observationperiod for the 60 μg and 90 μg (and 120 μg) TRE doses, whereas in the 30μg dose group the hemodynamic changes had just returned to baselinewithin this period. Even at the highest doses, TRE had only minoreffects on systemic arterial pressure (FIG. 8). Cardiac output wasincreased to a maximum of 106.8±3.2% (30 μg), 122.9±4.3% (60 μg),114.3±4.8% (90 μg) and 111.3±3.9% (120 μg TRE). The areas between theresponse curves after placebo versus TRE inhalation were calculated forPVR, PAP, SVR and SAP (FIG. 9). Areas between the curves for PVR werenot significantly different for 30 μg, 60 μg and 90 μg TRE, a nearlymaximal effect on PVR was already observed with 30 μg TRE. Effects onPAP and SAP were small and did not show a dose-response relationship.Gas exchange was not affected at doses up to 90 μg TRE, but arterialoxygen saturation was significantly decreased at a dose of 120 μg TRE inall 3 patients. Further dose increments were omitted due to this sideeffect and severe headache in one patient.

Again, bad taste of the TRE aerosol was reported by most patients. Otherside effects were flushing (n=1; 30 μg TRE), mild transient cough (n=3;60 μg TRE), mild transient bronchoconstriction that resolved after oneinhalation of fenoterol (n=1; 30 μg TRE), moderate bronchoconstrictionthat resolved after one inhalation of fenoterol (n=1; 120 μg TRE), andsevere headache (n=1; 120 μg TRE). The bad taste, thebronchoconstriction and the drop in SaO2 was attributed to metacresol inthe original TRE solution. With the use of a metacresol-free solution ofTRE (University Hospital Giessen, Germany; produced according to themanufacturer's protocol) in the following study, these side effects didno longer occur.

Study iii) was performed with metacresol-free TRE solution, having nospecific taste and smell. A total of 48 patients were enrolled. Thisstudy aimed at the reduction of inhalation time and aerosol volumeneeded for pulmonary drug delivery. A modified Optineb inhalation devicewas programmed to produce a constant amount of aerosol during repeatablepulses of aerosol generation. With this device, treprostinil could besafely utilized up to a concentration of 2000 μg/ml without considerableside effects. No relationship of number or type of side effects to TREconcentration was observed. Reported side effects were mild transientcough (n=6), mild headache (n=2) and mild jaw pain (n=1).

The reduction of PVR and PAP was comparable between all groups (FIG.10). TRE inhalation reduced PVR to 76.3±5.6% (18 pulses, 100 μg/ml),72.9±4.9% (9 pulses, 200 μg/ml), 71.2±6.0% (3 pulses, 600 μg/ml),77.4±4.5% (2 pulses, 1000 μg/ml) and 80.3±5.2% (1 pulse, 2000 μg/ml).PAP was reduced to 84.2±4.5% (18 pulses, 100 μg/ml), 84.2±4.1% (9pulses, 200 μg/ml), 81.1±4.1% (3 pulses, 600 μg/ml), 86±4% (2 pulses,1000 μg/ml) and 88±5.4% (1 pulse, 2000 μg/ml). Cardiac output wasmoderately increased in all groups, whereas systemic arterial pressurewas not significantly affected.

The areas between the curves (ABC) for changes in hemodynamic andgas-exchange parameters after inhalation of 15 μg TRE versus placebowere calculated for an observation time of 120 minutes (FIG. 11). TheABC for both PVR and PAP was comparable between all groups.

Pharmakokinetic results from study ii): Peak plasma concentrations oftreprostinil were found 10-15 minutes after inhalation. Maximaltreprostinil plasma concentrations (C_(max)) for the 30 μg, 60 μg, 90 μgand 120 μg doses were 0.65±0.28 ng/ml (n=4), 1.59±0.17 ng/ml (n=4), 1.74ng/ml (n=1) and 3.51±1.04 ng/ml (n=2), respectively (mean±SEM; FIG. 12).

Discussion:

These studies investigated whether i) the acute effects of inhaledtreprostinil would be comparable to or possibly advantageous overinhaled iloprost in pulmonary hypertensive patients, ii) the inhaledprostanoid dose might be increased without substantial local or systemicside effects, and iii) if the time of inhalation, which is 6-12 minutesfor iloprost, could be reduced significantly by increasing theconcentration of treprostinil aerosol.

The patient population in these studies included different forms ofprecapillary pulmonary hypertension. All these patients had a need fortherapy of pulmonary hypertension and reflected the typical populationof a pulmonary hypertension center. No major differences in patientcharacteristics or hemodynamic baseline values existed between thedifferent groups (table 3).

In study i) it was shown that the inhalation of treprostinil andiloprost in similar doses resulted in a comparable maximum pulmonaryvasodilatory effect. However, marked differences in the response profilewere noted. The onset of the pulmonary vasodilatory effect of inhaledtreprostinil was delayed compared to iloprost, but lasted considerablylonger, with the PVR decrease continuing beyond the one-hour observationperiod. Although the average dose of treprostinil was higher than theiloprost dose, no systemic effects were noted after treprostinilinhalation, whereas flush and transient SAP decrease, accompanied bymore prominent cardiac output increase, occurred after iloprostinhalation. Such side effects were more prominent than in previousstudies with inhaled iloprost. This may have been caused by the factthat the iloprost dose used in this study was 50% higher than therecommended single inhalation dose (5 μg) and that the precedingtreprostinil inhalation may have added to the systemic side effectscaused by the iloprost inhalation. Surprisingly, with TRE there was nosuch systemic side effect, although the average effect on PVR was aspotent as with iloprost.

This study used a cross-over design in order to minimize the effects ofinter-individual differences in response to prostanoids. The shortobservation period of 1 hour was used to avoid an uncomfortably longcatheter investigation. As a study limitation, the short observationinterval may have caused carryover effects of the first to the secondperiod as suggested by FIG. 5. However, this still allowed for theinterpretation of the study, that both drugs are potent pulmonaryvasodilators and that treprostinil effects are significantly sustainedcompared to the iloprost effects.

The longer duration of action and the virtual absence of side effects(except the bitter taste of treprostinil aerosol, later attributed tometacresol) encouraged increasing the applied treprostinil dose in studyii). Observation time was extended to 3 hours to obtain precisepharmacodynamic data. Inhaled treprostinil resulted in a strongpulmonary vasodilation that outlasted the observation time of 3 hourswhen compared to placebo inhalation. Surprisingly, inhaled treprostinilwas tolerated in doses up to 90 μg.

Study iii) successfully demonstrated that the inhalation time could bereduced to literally one single breath of 2000 μg/ml treprostinilsolution, thereby applying a dose of 15 μg. This drug administrationwith a single breath induced pulmonary vasodilation for longer than 3hours compared to placebo inhalation. Side effects were minor, of lowfrequency and not related to drug concentration. It was a surprisingfinding that such high concentrations of treprostinil were so welltolerated.

Conclusion:

Inhaled treprostinil can be applied in high doses (up to 90 μg) with aminimal inhalation time. Inhaled treprostinil exerts high pulmonaryselectivity and leads to a long-lasting pulmonary vasodilation.

Although the foregoing refers to particular preferred embodiments, itwill be understood that the present invention is not so limited. It willoccur to those of ordinary skill in the art that various modificationsmay be made to the disclosed embodiments and that such modifications areintended to be within the scope of the present invention.

All of the publications, patent applications and patents cited in thisspecification are incorporated herein by reference in their entirety.

What is claimed is:
 1. A method of treating pulmonary hypertensioncomprising: administering by inhalation to a human suffering frompulmonary hypertension a therapeutically effective single event dose ofa formulation comprising from 200 to 1000 μg/ml of treprostinil or apharmaceutically acceptable salt thereof with a pulsed ultrasonicnebulizer that aerosolizes a fixed amount of treprostinil or apharmaceutically acceptable salt thereof per pulse, said pulsedultrasonic nebulizer comprising an opto-acoustical trigger which allowssaid human to synchronize each breath to each pulse, saidtherapeutically effective single event dose comprising from 15 μg to 90μg of treprostinil or a pharmaceutically acceptable salt thereofdelivered in 1 to 18 breaths.
 2. The method of claim 1, wherein theformulation comprises 600 μg/ml of the treprostinil or itspharmaceutically acceptable salt thereof.
 3. The method of claim 1,wherein the single event dose is not repeated for a period of at least 3hours.
 4. The method of claim 1, wherein the single event dose producesa peak plasma concentration of treprostinil about 10-15 minutes afterthe single event dose.
 5. The method of claim 1, wherein the fixedamount of treprostinil or its pharmaceutically salt for each breathinhaled by the human comprises at least 5 μg of treprostinil or itspharmaceutically acceptable salt.
 6. The method of claim 2, wherein thefixed amount of treprostinil or its pharmaceutically salt for eachbreath inhaled by the human comprises at least 5 μg of treprostinil orits pharmaceutically acceptable salt.
 7. The method of claim 1, whereinthe single event dose is inhaled in 3-18 breaths by the human.
 8. Themethod of claim 6, wherein the single event dose is inhaled in 3-18breaths by the human.
 9. The method of claim 6, wherein the single eventdose is not repeated for a period of at least 3 hours.